UNIVERSITI PUTRA MALAYSIA PRODUCTION OF …psasir.upm.edu.my/4815/1/FBSB_2005_2.pdf · immunoblot terhadap interaksi di antara antigen dan antibodi menunjukkan bahawa antibodi anti-F

UNIVERSITI PUTRA MALAYSIA

PRODUCTION OF RECOMBINANT ENVELOPE PROTEINS OF NEWCASTLE DISEASE VIRUS IN ESCHERICHIA COLI AND ANALYSIS

OF THEIR IMMUNOLOGICAL PROPERTIES

WONG SING KING

FBSB 2005 2

1

PRODUCTION OF RECOMBINANT ENVELOPE PROTEINS OF

NEWCASTLE DISEASE VIRUS IN ESCHERICHIA COLI AND ANALYSIS OF

THEIR IMMUNOLOGICAL PROPERTIES

WONG SING KING

DOCTOR OF PHILOSOPHY

UNIVERSITI PUTRA MALAYSIA

2005

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By

WONG SING KING

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfilment of the Requirements for the Degree of Doctor of Philosophy

December 2004

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The fear of the LORD LORD LORD LORD is the beginning of wisdom,

and knowledge of the Holy OneHoly OneHoly OneHoly One is understanding.

~ Proverbs~ Proverbs~ Proverbs~ Proverbs

4

To my dearest father and mother

for their infinite love, care and support.

I owe them everything I have today.

To my beloved brothers and sister.

Also to my relatives and friends.

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of

the requirements for the degree of Doctor of Philosophy




By

WONG SING KING

December 2004

Chairman : Professor Datin Khatijah Mohd. Yusoff, PhD

Faculty : Biotechnology and Biomolecular Sciences

Newcastle disease virus (NDV) is the causative agent of the Newcastle disease (ND) that

remains as a major threat to the world poultry industry. The virus belongs to the family

Paramyxoviridae and genus Avulavirus, infects more than 236 avian species, and causes

up to 100% morbidity and mortality in susceptible birds. The viral envelope proteins,

haemagglutinin-neuraminidase (HN) and fusion (F) proteins have been shown to play

key roles in triggering the host immune responses. In order to study the immunological

properties of the recombinant HN and F proteins, the HN and F genes of the Malaysian

viscerotropic-velogenic NDV strain AF2240 were obtained through reverse

transcription-polymerase chain reaction (RT-PCR) and cloned into the Pichia pastoris,

Saccharomyces cerevisiae and Escherichia coli expression vectors.

A number of eight recombinant plasmids were constructed, namely pPICZαA/HN and

pPICZαA/F (P. pastoris system), pYES2α/HN and pYES2α/F (S. cerevisiae system),

and pRSETA/HN, pRSETB/F, pET-43.1a/HN and pET-43.1a/F (E. coli system). The

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recombinant plasmids were used to transform respective host cells, which were then

induced for the production of the recombinant HN and F proteins. However, there was

no protein expression observed in the recombinant P. pastoris and S. cerevisiae cells.

Whereas, the bacterial hosts were found expressing the recombinant HN and F proteins

(from the pRSETA/HN and pRSETB/F plasmids respectively), and the NusA fusion

proteins, NusA-HN and NusA-F (from the pET-43.1a/HN and pET-43.1a/F plasmids

respectively). The recombinant HN and F proteins were produced as insoluble inclusion

bodies (IB) while the NusA-HN and NusA-F proteins were expressed in soluble form in

E. coli.

The recombinant proteins were purified and used to immunise specific pathogen-free

(SPF) chickens. ELISA results revealed that the insoluble and urea-solubilised inclusion

bodies of the recombinant HN and F proteins, and the soluble NusA-HN and NusA-F

proteins stimulated the production of antibodies that detect NDV. Among these antigens,

the urea-solubilised HN IB appeared to induce the highest antibody titers. However, the

chicken antibodies failed to neutralise the viral activities as shown in the tests such as

haemagglutination inhibition (HI), neuraminidase inhibition (NI) and haemolysis

inhibition (HLI). This explains the susceptibility of the immunised flocks to NDV

infection upon the viral challenge. Despite of the presence of antibodies to NDV, none

of the immunised chicken was protected against the viral challenge. Immunoblotting

analysis on the interactions between the antigens and antibodies revealed that the anti-F

antibodies did not bind to the denatured viral F glycoprotein, neither the anti-NDV

serum detect the recombinant F protein. However, the anti-HN antibodies showed

positive signals when used to probe the denatured viral HN glycoprotein, and in return,

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the anti-NDV serum detected the recombinant HN protein. This finding indicates the

potential application of the E. coli produced HN protein as antigen for the detection of

NDV antibody.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Doktor Falsafah

PENGHASILAN PROTEIN SELAPUT REKOMBINAN VIRUS PENYAKIT

NEWCASTLE DALAM ESCHERICHIA COLI DAN ANALISIS CIRI-CIRI

IMUNOLOGINYA

Oleh

WONG SING KING

Disember 2004

Pengerusi : Profesor Datin Khatijah Mohd. Yusoff, PhD

Fakulti : Bioteknologi dan Sains Biomolekul

Virus penyakit Newcastle (NDV) merupakan agen penyebab bagi penyakit Newcastle

(ND) yang masih menjadi ancaman utama kepada industri ayam sedunia. Virus ini

berada dalam famili Paramyxoviridae dan genus Avulavirus. Ia menjangkiti lebih

daripada 236 spesis burung dan boleh menyebabkan morbiditi dan mortaliti setinggi

100% dalam burung-burung yang terjangkit. Protein selaput virus, iaitu protein

hemaglutinin-neuraminidase (HN) and protein pertaupan (F) telah ditunjukkan

memainkan peranan yang penting dalam merangsangkan tindakbalas keimunan. Untuk

mengkaji sifat-sifat imunologik protein rekombinan HN dan F, maka gen-gen HN and F

bagi strain AF2240 NDV Malaysia yang viscerotropik-velogenik diperolehi menerusi

transkripsi terbalik-tindakbalas rantaian polimerase (RT-PCR) dan seterusnya diklon ke

dalam vektor pengekspresan Pichia pastoris, Saccharomyces cerevisiae and Escherichia

coli.

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Sejumlah lapan plasmid rekombinan telah dibina, iaitu pPICZαA/HN dan pPICZαA/F

(sistem P. pastoris), pYES2α/HN dan pYES2α/F (sistem S. cerevisiae), serta

pRSETA/HN, pRSETB/F, pET-43.1a/HN dan pET-43.1a/F (sistem E. coli). Plasmid

rekombinan ini digunakan untuk mentransfomkan sel perumah masing-masing, yang

seterusnya diaruh untuk penghasilan protein rekombinan HN dan F. Namun, tiada

pengekspresan protein diperhatikan dalam sel rekombinan P. pastoris dan S. cerevisiae.

Manakala perumah bakteria telah didapati bahawa ianya mengekspreskan protein

rekombinan HN dan F (dari plasmid pRSETA/HN and pRSETB/F masing-masing), serta

protein yang bergabungan dengan NusA, iaitu NusA-HN dan NusA-F (dari plasmid

pET-43.1a/HN dan pET-43.1a/F masing-masing). Protein-protein rekombinan HN dan F

dihasilkan sebagai badan inklusi (IB) yang tak terlarutkan, sementara protein NusA-HN

dan NusA-F telah diekspreskan dalam bentuk terlarutkan dalam E. coli.

Protein rekombinan ini ditulenkan dan seterusnya digunakan untuk mengimunkan ayam

bebas patogen spesifik (SPF). Keputusan ELISA menunjukkan bahawa badan inklusi tak

terlarutkan dan terlarutkan urea bagi protein-protein rekombinan HN dan F, serta protein

NusA-HN dan NusA-F yang terlarutkan berjaya merangsangkan penghasilan antibodi

yang mengesan NDV. Antara antigen ini, badan inklusi HN yang dilarutkan dalam urea

telah mencetuskan titer antibodi yang tertinggi. Walau bagaimanapun, antibodi ayam ini

gagal menuetralkan aktiviti virus sebagaimana yang ditunjukkan dalam ujian seperti

perencatan hemaglutinasi (HI), perencatan neuraminidase (NI) dan perencatan hemolisis

(HLI). Ini menjelaskan keterjangkitan NDV terhadap ayam yang telah diimunkan itu

setelah dicabar dengan virus tersebut. Walaupun terdapatnya antibodi terhadap NDV,

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namun tiada ayam yang diimunkan itu terlindung daripada cabaran virus. Analisis

immunoblot terhadap interaksi di antara antigen dan antibodi menunjukkan bahawa

antibodi anti-F tidak mengenali glikoprotein F virus yang nyahasli, begitu juga dengan

serum anti-NDV yang tidak mengesan protein rekombinan F. Namun, antibody anti-HN

mengikat glikoprotein HN virus yang nyahasli, begitu juga dengan serum anti-NDV

yang dapat mengesan protein rekombinan HN. Penemuan ini menunjukkan potensi

kegunaan protein HN yang dihasilkan dalam E. coli ini sebagai antigen dalam

pengesanan antibodi NDV.

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ACKNOWLEDGEMENTS

All the glories and thanksgivings to God almighty in the highest.

My deepest gratitude to my supervisors, Prof. Datin Dr. Khatijah Mohd. Yusoff,

Assoc. Prof. Dr. Tan Wen Siang, Dr. Tan Chon Seng and Assoc. Prof. Dr. Abdul

Rahman Omar for their invaluable guidance, help and patience throughout the project,

and for the critical review in the completion of this thesis.

I am indeed indebted to my seniors in the Virology Lab 143: Omeima, Alan,

Subha, Chui Fong, Putery, Vijay, Wei Hong and Chiew Ling for sharing their

knowledge and experience with me.

I appreciate the friendship and assistance from all my labmates: Pria, Amir, Kok

Lian, Wai Ling, Tang, Majid, Dr. Azri, Geok Hun, Lalita, Eddie, Thong Chuan, kak

Raha, Riha, Wawa, Sharifah, Su, Rafidah, Zul, Onie, Eni, Firoozeh, Swee Tin, Kah Fai,

Andrew, Shirley Foo, Taznim, Salwa and Yan Peng.

My special thanks to Ina who had shared my burden and worked restlessly in

protein purification and field trial. I am deeply grateful to her commitment and

assistance.

I would like to express my sincere gratitude to Assoc. Prof. Dr. Abdullah Sipat,

Assoc. Prof. Dr. Che Nyonya Abdul Razak, Prof. Abu Bakar Salleh, Assoc. Prof. Dr.

Raja Noor Zaliha Raja Abdul Rahman, Assoc. Prof. Dr. Tong Chow Chin and Assoc.

Prof. Dr. Raha Abdul Rahim for their approval for me to access the facilities in their

laboratories.

I wish to extend my appreciation to everyone, although not individually named

here, who had contributed directly or indirectly to my project and thesis.

This study was supported by IRPA grants of The Ministry of Science,

Technology and Environment of Malaysia.

Last but not least, I want to take the opportunity to thank my parents, brothers

and sister for their endless love, care and encouragement.

Without all of you, it would not be possible for me to complete my project and

thesis. May God bless you all for your kindness.

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I certify that an Examination Committee met on 31st December 2004 to conduct the final

examination of Wong Sing King on his Doctor of Philosophy thesis entitled “Production

of Recombinant Envelope Proteins of Newcastle Disease Virus (NDV) in Escherichia

coli and Analysis of their Immunological Properties” in accordance with Universiti

Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia

(Higher Degree) Regulations 1981. The Committee recommends that the candidate be

awarded the relevant degree. Members of the Examination Committee are as follows:

ABDUL MANAF ALI, PhD

Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

RAJA NOOR ZALIHA RAJA ABDUL RAHMAN, PhD

Associate Professor



(Member)

SITI SURI ARSHAD, PhD

Associate Professor

Faculty of Veterinary Medicine


(Member)

JIMMY KWANG, PhD

Professor

Temasek Life Sciences Laboratory

National University of Singapore

(Independent Examiner)

_________________________________

GULAM RUSUL RAHMAT ALI, PhD

Professor/Deputy Dean

School of Graduate Studies


Date:

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This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted

as fulfillment of the requirement for the degree of Doctor of Philosophy. The members

of the Supervisory Committee are as follows:

DATIN KHATIJAH MOHD. YUSOFF, PhD

Professor



(Chairman)

TAN WEN SIANG, PhD

Associate Professor



(Member)

TAN CHON SENG, PhD

Senior Researcher

Biotechnology Unit

Malaysia Agriculture Research and Development Institute

(Member)

ABDUL RAHMAN OMAR, PhD

Associate Professor

Faculty of Veterinary Medicine


(Member)

________________________

AINI IDERIS, PhD

Professor/Dean

School of Graduate Studies


Date:

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DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and

citations which have been duly acknowledged. I also declare that it has not been

previously or concurrently submitted for any other degree at UPM or other institutions.

__________________

WONG SING KING

Date:

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TABLE OF CONTENTS

Page

DEDICATION ii

ABSTRACT iii

ABSTRAK vi

ACKNOWLEDGEMENTS ix

APPROVAL x

DECLARATION xii

LIST OF TABLES xvii

LIST OF FIGURES xviii

LIST OF ABBREVIATIONS xxi

CHAPTER

1 INTRODUCTION 1.1

2 LITERATURE REVIEW 2.1

2.1 Newcastle Disease 2.1

2.1.1 History 2.1

2.1.2 Host Range 2.2

2.1.3 Pathotypes 2.3

2.1.4 Diagnosis 2.4

2.1.5 Host Immune Response 2.6

2.2 Newcastle Disease Virus (NDV) 2.7

2.2.1 Classification 2.7

2.2.2 Morphology 2.8

2.2.3 Virion Composition 2.8

2.2.4 Viral Genome 2.10

2.2.5 Nucleocapsid Protein (NP) 2.10

2.2.6 Phosphoprotein (P) and Non-Structural Proteins

(V and W) 2.12

2.2.7 Large (L) Protein 2.12

2.2.8 Matrix (M) Protein 2.13

2.2.9 Haemagglutinin-Neuraminidase (HN) Protein 2.13

2.2.10 Fusion (F) Protein 2.17

2.3 Virus Entry and Replication 2.19

2.4 NDV Strain AF2240 2.22

2.5 Immunological Properties of the Recombinant HN and F

Proteins Expressed in Various Expression Systems 2.24

2.6 Escherichia coli Expression System 2.27

2.6.1 Expression Vectors 2.27

2.6.2 Strategies for Protein Expression 2.31

2.6.2.1 Intracellular Expression 2.31

2.6.2.2 Extracytoplasmic Expression 2.33

2.6.3 Gene Fusion Systems 2.34

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2.7 Yeast Expression Systems 2.38

2.7.1 Saccharomyces cerevisiae 2.38

2.7.1.1 Yeast Vectors 2.40

2.7.1.2 Secretion of Heterologous Proteins 2.43

2.7.2 Pichia pastoris 2.47

3 MATERIALS & METHODS 3.1

3.1 Molecular Cloning Methods 3.1

3.1.1 General Procedures 3.1

3.1.2 NDV Strain AF2240 3.2

3.1.3 Virus Propagation and Purification 3.2

3.1.4 Virus Titration by Haemagglutination (HA) Test 3.3

3.1.5 Viral Genomic RNA Isolation 3.4

3.1.6 Primer Design 3.4

3.1.7 Reverse Transcription-Polymerase Chain Reaction

(RT-PCR) 3.5

3.1.8 Cloning of HN and F Genes into In Vitro, Yeast and

Bacterial Expression Vectors 3.7

3.1.8.1 Overview of the Expression Vectors 3.7

3.1.8.2 Preparation of Escherichia coli Competent

Cells 3.7

3.1.8.3 Cloning of HN and F Genes into pPICZαA

Vector 3.9

3.1.8.4 Heat-Shock Transfomation of Escherichia coli 3.10

3.1.8.5 Plasmid Extraction and Restriction Enzyme

Analysis 3.11

3.1.8.6 Nucleotide Sequencing of the HN and F Gene

Inserts 3.14

3.1.8.7 Subcloning of HN and F Genes into Other

Expression Vectors 3.15

3.1.9 Electroporation Transformation of Yeast 3.17

3.1.10 Direct PCR Analysis of the Yeast Transformants 3.20

3.2 Protein Expression Methods 3.21

3.2.1 In Vitro Transcription and Translation of the HN and F

Genes 3.21

3.2.2 SDS-Polyacryamide Gel Electrophoresis (SDS-PAGE) 3.22

3.2.3 Protein Expression in Pichia pastoris 3.23

3.2.4 Protein Expression in Saccharomyces cerevisiae 3.25

3.2.5 Protein Expression in Escherichia coli BL21-SI 3.26

3.2.6 Protein Expression in Escherichia coli Origami B(DE3) 3.27

3.2.7 Western Blot Analysis 3.28

3.2.8 Solubility Fractionation of the Recombinant Proteins 3.29

3.3 Protein Purification Methods 3.30

3.3.1 Isolation of Recombinant Protein Inclusion Bodies (IB) 3.30

3.3.2 Purification of Soluble Recombinant Proteins 3.31

3.3.3 Protein Quantitation 3.33

3.3.3.1 The Bradford Method 3.33

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3.3.3.2 Densitometry Method 3.33

3.4 Immunological Methods 3.34

3.4.1 Immunisation of the Specific Pathogen-Free (SPF)

Chickens 3.34

3.4.2 Challenge Study 3.35

3.4.3 Enzyme-Linked Immunosorbent Assay (ELISA) 3.35

3.4.4 Immunoblotting 3.35

3.4.5 Haemagglutination Inhibition (HI) Test 3.36

3.4.6 Neuraminidase Inhibition (NI) Test 3.37

3.4.7 Haemolysis Inhibition (HLI) Test 3.37

4 RESULTS 4.1

4.1 Amplification of the HN and F Genes 4.1

4.2 Cloning and Expression in Pichia pastoris System 4.3

4.2.1 Cloning of the HN and F Genes into pPICZαA Vector 4.5

4.2.2 PCR Screening of Yeast Transformants 4.5

4.2.3 Partial Terminal Nucleotide Sequencing of the HN and

F Gene Inserts 4.8

4.2.4 Protein Expression in Pichia pastoris 4.11

4.3 Cloning and Expression in In Vitro Transcription and

Translation System 4.13

4.3.1 Subcloning of the HN and F Genes into the pCITE-2a

Vector 4.14

4.3.2 In Vitro Transcription and Translation 4.14

4.4 Cloning and Expression in Saccharomyces cerevisiae System 4.18

4.4.1 Construction of the Recombinant pYES2α/HN and

pYES2α/F Plasmids 4.18

4.4.2 Direct PCR Analysis of Yeast Transformants 4.20

4.4.3 Protein Expression in Saccharomyces cerevisiae 4.22

4.5 Cloning and Expression in Bacterial Systems 4.22

4.5.1 Subcloning of the HN and F Genes into the pRSET

Vectors 4.22

4.5.2 Expression of the Recombinant HN and F Proteins in

Escherichia coli BL21-SI 4.23

4.5.3 Subcloning of the HN and F Genes into the pET-43.1a

Vectors 4.26

4.5.4 Expression of the Recombinant NusA-HN and NusA-F

Proteins in Escherichia coli Origami B(DE3) 4.29

4.5.5 Solubility of the Recombinant Proteins 4.34

4.5.6 Isolation and Solubilisation of the Recombinant HN

and F Inclusion Bodies 4.34

4.5.7 Purification of the Soluble NusA-HN and NusA-F

Proteins 4.38

4.5.8 Quantitation of the Recombinant Proteins 4.41

4.6 Antibody Production in Chickens 4.43

4.7 Viral Challenge 4.48

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4.8 Immunoblotting Analysis of the Recombinant Proteins with the

Chicken Sera 4.48

4.9 Haemagglutination Inhibition (HI), Neuraminidase Inhibition

(NI) and Haemolysis Inhibition (HLI) Tests 4.54

5 DISCUSSION 5.1

5.1 Recombinant Protein Expression in Yeasts 5.1

5.2 Recombinant Protein Expression in Escherichia coli 5.4

5.3 Protein Solubility 5.6

5.4 Protein Purification 5.11

5.5 Immunogenic Properties of the Recombinant HN and F

Proteins 5.14

6 CONCLUSION 6.1

BIBLIOGRAPHY R.1

APPENDICES A.1

BIODATA OF THE AUTHOR B.1

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LIST OF TABLES

Table Page

2 Protein and Peptide Fusion Partners for Purification and Detection of

the Recombinant Proteins 2.37

3.1 Oligonucleotide Primers used in Amplification of the HN and F Genes 3.6

3.2 Properties of the Expression Vectors used in this study 3.8

3.3 Restriction Enzymes and the Reaction Buffers 3.13

4.1 Expression Levels and Solubility Ratios of the Recombinant HN and F

Proteins 4.35

4.2 ELISA Titers of the Chicken Serum Samples 4.45

4.3 Immunoblotting Analysis of the Recombinant HN and F Proteins with

the Chicken Sera 4.50

4.4 Haemagglutination Inhibition (HI), Neuraminidase Inhibition (NI) and

Haemolysis Inhibition (HLI) Tests on the Chicken Sera 4.55

5.1 Predicted Solubilities of Carrier, Target and Fusion Proteins 5.9

5.2 Antigenic Count of the HN Protein using the EMBOSS Antigenic

Program 5.20

5.3 Antigenic Count of the F Protein using the EMBOSS Antigenic

Program 5.21

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LIST OF FIGURES

Figure Page

1 Newcastle Disease in the World in 2002 1.2

2.1 Schematic Diagram of NDV Virion 2.9

2.2 Transcription and Replication of the NDV Genome 2.11

2.3 X-Ray Structure of the NDV HN Protein 2.16

2.4 X-Ray Structure of the NDV F Protein 2.18

2.5 A Model Pathway of NDV-Host Membrane Fusion 2.20

2.6 Nucleotide and Deduced Amino Acid Sequences of HN Gene of NDV

AF2240 2.23

2.7 Nucleotide and Deduced Amino Acid Sequences of F Gene of NDV

AF2240 2.25

2.8 Schematic of the Promoter and Regulatory Systems for Recombinant

Gene Expression in E. coli 2.30

2.9 Comparison of Yeast and Mammalian N-Linked Glycosylation 2.45

2.10 Gene Insertion and Transplacement into Pichia Genome 2.49

3.1 Flow Chart of the Construction of the Recombinant Plasmids carrying

the HN Gene 3.18

3.2 Flow Chart of the Construction of the Recombinant Plasmids carrying

the F Gene 3.19

4.1 Cloned Regions of the HN and F Genes 4.2

4.2 RT-PCR Amplification of the Extracellular Domains Coding Regions

of the HN and F Genes of NDV Strain AF2240 4.4

4.3 EcoRI-KpnI Restriction Digestion of the pPICZαA/HN and pPICZαA/F

Plasmids 4.6

4.4 Linearisation of the pPICZαA/HN and pPICZαA/F Plasmids 4.7

4.5 Colony PCR on the Recombinant Yeast 4.9

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4.6 5’ End Terminal Sequencing of the pPICZαA/F and pPICZαA/HN

Plasmids 4.10

4.7 3’ End Terminal Sequencing of the pPICZαA/F and pPICZαA/HN

Plasmids 4.12

4.8 EcoRI-KpnI Restriction Digestion of the pCITE-2a/HN and pCITE-2a/F

Plasmids 4.15

4.9 Linearisation of the pCITE-2a/HN and pCITE-2a/F Plasmids 4.16

4.10 In Vitro Translation of the HN and F Polypeptides 4.17

4.11 Construction of the pYES2α/HN and pYES2α/F Plasmids 4.19

4.12 BamHI-HindIII Restriction Digestion of the pYES2α/F and pYES2α/HN

Plasmids 4.21

4.13 EcoRI-KpnI Restriction Digestion of the pRSETA/HN and pRSETB/F

Plasmids 4.24

4.14 Time-Course Expression of the Recombinant HN Protein in E. coli

BL21-SI 4.25

4.15 Time-Course Expression of the Recombinant F Protein in E. coli

BL21-SI 4.27

4.16 Western Blot Analysis of the Recombinant HN and F Proteins with

Anti-His Monoclonal Antibody 4.28

4.17 EcoRI-KpnI Restriction Digestion of the pET-43.1a/HN and pET-43.1a/F

Plasmids 4.30

4.18 Time-Course Expression of the Recombinant NusA-F Protein in E. coli

Origami B(DE3) 4.31

4.19 Time-Course Expression of the Recombinant NusA-HN Protein in E. coli

Origami B(DE3) 4.32

4.20 Western Blot Analysis of the NusA-HN and NusA-F Proteins with

Anti-His Monoclonal Antibody 4.33

4.21 Solubility of the Recombinant HN and F Proteins Expressed in E. coli

BL21-SI 4.36

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4.22 Solubility of the NusA-HN and NusA-F Proteins Expressed in E. coli

Origami B(DE3) 4.37

4.23 Isolation of the Recombinant HN Inclusion Body (IB) 4.39

4.24 Isolation of the Recombinant F Inclusion Body (IB) 4.40

4.25 Affinity Purification of the NusA, NusA-HN and NusA-F Proteins 4.42

4.26 Antibody Titers of the Chickens Immunised with the Recombinant HN

and F Proteins 4.44

4.27 Immunoblotting Analyses using the Chicken Sera against the Recombinant

HN and F Proteins 4.51

4.28 Immunoblotting Analyses using the Chicken Sera against the NusA-HN

and NusA-F Proteins 4.52

4.29 Immunoblotting Analyses using the Chicken Sera against NDV and the

NusA Proteins 4.53

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LIST OF ABBREVIATIONS

A adenine

ADH1 alcohol dehydrogenase 1

ADH2 alcohol dehydrogenase II

AGR4 argininosuccinate lyase

Ala (A) alanine

AMV avian myeloblastosis virus

AOX alcohol oxidase

APMV-1 avian paramyxovirus type-1

APS ammonium persulfate

Arg (R) arginine

ARS autonomously replicating sequence

Asn (N) asparagine

Asp (D) aspartic acid

ATP adenosine-5’-triphosphate

ATPase adenosine triphosphatase

BCIP bromochloroindolyl phosphate

BiP heavy chain binding protein

BMGY buffered glycerol-complex medium

BMMY buffered methanol-complex medium

BSA bovine serum albumin

C cytosine

cDNA complementary DNA

CEF chicken embryo fibroblast

CITE cap-independent translation enhancer

CMI cell-mediated immunity

CMV cytomegalovirus

CTP cytidine-5’-triphosphate

CYC1 iso-1-cytochrome c

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Cys (C) cysteine

dATP deoxyadenosine-5’-triphosphate

dCTP deoxycytidine-5’-triphosphate

ddNTPs dideoxyribonucleotides or dideoxyribonucleoside-5’-

triphosphates

DEPC diethylpyrocarbonate

dGTP deoxyguanosine-5’-triphosphate

dH2O distilled water

DNA deoxyribonucleic acid

dNTPs deoxyribonucleotides or deoxyribonucleoside-5’-triphosphates

DTT dithiothreitol

dTTP thymidine-5’-triphosphate

EDTA ethylenediaminetetraacetic acid

EF-Tu elongation factor Tu

EID50 mean egg infectious dose

ELISA enzyme-linked immunosorbent assay

ER endoplasmid reticulum

F fusion (protein)

FPV fowlpox virus

G guanine

g gravity

Gal galactose

GAP glyceraldehydes-3-phoshate dehydrogenase

GlcNAc N-acetyl-glucosamine

Gln (Q) glutamine

Glu (E) glutamic acid

Gly (G) glycine

gor gluthathione oxido-reductase

GRAS generally regarded as safe

GST glutathione S-transferase

GTP guanosine-5’-triphosphate

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UNIVERSITI PUTRA MALAYSIA PRODUCTION OF …psasir.upm.edu.my/4815/1/FBSB_2005_2.pdf · immunoblot terhadap interaksi di antara antigen dan antibodi menunjukkan bahawa antibodi anti-F

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